This invention relates generally to a stabilized form of enzyme creatine kinase (CK) which will be stable in serum and will adequately mimic CK in human samples for assay by various analytical procedures for determination of CK in human serum.
It is well known that the instability of many sulfhydryl (SH) containing enzymes are mainly due to the irreversible modification of the reactive SH group. CK is an example of such an enzyme that is present in human serum and is commonly measured for the purpose of diagnosing myocardial infarction. In the application of various assays for CK in human serum for this purpose, it is required to have available controls, calibrators and other reference materials which contain a known amount or concentration of this enzyme (CK). It is a general objective to achieve maximum stability of all the parameters in such reference materials, and it is known that CK is one of the less stable compounds added to such reference materials, by virtue of its reactive SH group. Therefore, the present invention aims to achieve a modified form of CK that will have increased stability in reference materials, specifically increased stability in the lyophilized material after reconstitution by addition of diluent. At the same time it is essential that the stabilized CK used in such products will adequately mimic the CK present in various human serum samples, for different analytical procedures used to determine CK.
In order to preserve the enzyme activity of CK and other SH-containing enzymes traditionally one or more thiol-compounds are added to the CK solution at high concentration. This approach cannot be used in the present instance, because such thiol compounds will interfere with other analytical procedures for the determination of other constituents of such reference materials. An example is the determination of alkaline phosphatase, which shows reduced enzymatic activity in the presence of excess added thiol--due to complexation of the zinc contained in the active site of this enzyme.
Another approach to stabilizing--SH enzymes is through modification of the reactive--SH groups by some reagent. However, generally such reagents, e.g. iodoacetate, lead to irreversible reaction of the SH group and considerable or total loss in enzyme activity. This is also undesirable for the preparation of stabilized CK to be used in a reference material.
It has been found that the reaction of an organodisulfide, preferably cystine, gives a stabilized CK which is uniquely useful in meeting all the requirements of a stabilized CK to be included in reference serum. It is highly stable upon reconstitution and it is converted to the active form very quickly in solutions used to assay for CK. Thus, it perfectly mimics CK in human serum and serves as an effective reference material.